JP7384407B2 - Soil improvement material, its manufacturing method, and soil improvement method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Announced at the 2019 Research Results Presentation Meeting (Date: June 25, 2019)

The present invention relates to a soil improvement material, a method for producing the same, and a soil improvement method.

Continuously cultivating the same crop in the same place causes a disorder in which the crop grows poorly (so-called continuous cropping disorder). In order to prevent and improve this continuous cropping disorder, pesticides and materials containing microorganisms are known.

For example, Patent Document 1 discloses a plant pathogen control agent characterized by containing a culture, wet microbial cells, or dry microbial cells of specific Trichoderma genus bacteria.
However, pesticides and materials containing microorganisms are difficult to handle, such as complicated storage management, the need to use them immediately after preparation, and the need to prepare pesticides for each construction, and high production costs. There were problems such as the high price. Additionally, with liquid types such as microbial culture solutions, users may feel reluctant to handle live bacteria.

JP2013-255428A

An object of the present invention is to provide a soil improvement material that is easy to handle and inexpensive, and a method for producing the same. Another object of the present invention is to provide a soil improvement method using the soil improvement material of the present invention.

As a result of extensive research in order to solve the above problems, the present inventors found that the following invention met the above objects, leading to the present invention.

That is, the present invention relates to the following inventions.
<1> A method for producing a soil improvement material, comprising a first step of preparing a mixture containing a Trichoderma bacterium, a purified water cake, and a cellulose material, and a second step of culturing the Trichoderma bacterium in the mixture. .
<2> The manufacturing method according to <1> above, wherein the cellulose material is paper.
<3> The manufacturing method according to <1> or <2>, wherein the mass ratio of the cellulose material to the purified water cake in terms of solid content is 0.001 to 0.8.
<4> The Trichoderma bacterium used in the first step is a bacterium collected from the layer of soil in which the cellulose has been decomposed after being cultured in soil embedded with cellulose for three months or more. The manufacturing method according to any one of <3>.
<5> A soil improvement material manufactured by the manufacturing method according to any one of <1> to <4>.
<6> A soil improvement method for improving soil using the soil improvement material produced by any of the production methods of <1> to <4>.

According to the present invention, a soil improvement material that is easy to handle and inexpensive, and a method for producing the same are provided. Further, a soil improvement method using the soil improvement material of the present invention is provided.

This is an electrophoretic profile of bacterial PCR products. (a) Standard curve based on the relationship between cycle threshold and Trichoderma virens DNA concentration, (b) Melting curve (fluorescence versus temperature), and (c) Standard curve based on the relationship between cycle threshold and Trichoderma virens DNA concentration. It is a figure which shows a real-time amplification curve. It is a figure showing the result of the culture test of bacteria at each culture temperature. FIG. 3 is a diagram showing the number of spores versus culture time. It is a photograph showing the state of the Erlenmeyer flask in which Trichoderma bacteria were cultured and the state of the soil improvement material taken out after 40 days of culture. It is a photograph showing the results of a growth test of Komatsuna. They are photographs and diagrams showing the results of a growth test on tomato seedlings. It is a photograph showing the results of a growth test of radish. It is a photograph showing the results of a celery growth test. It is a figure showing the average value of the weight of salad greens cultivated for each soil. FIG. 2 is a diagram showing a photograph of Chinese cabbage on the 50th day of planting and the average weight of the edible portion of Chinese cabbage grown in each soil. It is a figure showing a photograph of carrots at the time of harvest and an average value of the weight of the edible portion of carrots grown in each soil. FIG. 2 is a diagram showing the average weight of edible parts of potatoes, bok choy, and broccoli grown in each soil. This is a photograph showing the results of cultivating tomato seedlings in soil that has not been inoculated with pathogens, contaminated soil that has not been treated with soil supplements (pathogen-inoculated soil), and contaminated soil that has been mixed with soil supplements (inoculated with pathogens + soil supplements). It is a diagram showing photographs of tomatoes grown in each soil 30 days after planting and the number of bacteria relative to the number of cultivation days. It is a photograph of soybean grown in each soil 30 days after planting, and a diagram showing the number of bacteria with respect to the number of cultivation days. These are photographs showing the results of the first and second continuous cultivation tests of Komatsuna.

The embodiments of the present invention will be described in detail below, but the explanation of the constituent elements described below is an example (representative example) of the embodiments of the present invention, and the present invention will be described below unless the gist thereof is changed. is not limited to the content.

<Method for producing soil improvement material of the present invention>
The present invention provides soil comprising: a first step of preparing a mixture containing Trichoderma bacteria, a purified water cake, and a cellulose material; and a second step of cultivating the Trichoderma bacteria in the mixture. The present invention relates to a method for manufacturing improved materials (hereinafter sometimes referred to as "the manufacturing method of the present invention").

The present inventors have discovered that the purified water cake is suitable as a culture carrier for bacteria of the genus Trichoderma, and that soil improvement materials obtained by culturing bacteria of the genus Trichoderma in the presence of cellulose materials using the purified water cake as a carrier can be continuously produced. It has been found that it has excellent effects on suppressing disorders and promoting crop growth.
Furthermore, it was found that the obtained soil improvement material could stably maintain the number of Trichoderma bacteria in the soil improvement material without complicated storage management. Therefore, the storage and management of soil improvement materials can be facilitated.

In addition, only a portion of the purified water cake used as one of the raw materials for the soil improvement material of the present invention is used for construction materials and road repair, and most of it is discarded in landfills. According to the present invention, the uses of the purified water cake can also be expanded.
Water purification cake tends to contain aluminum-based flocculants such as polyaluminum chloride used in the treatment process of water purification plants, and when used for soil improvement, phosphoric acid components in the soil that become nutrients for crops can be absorbed. was fixed by aluminum, which caused problems. However, the soil improvement material obtained by the production method of the present invention can increase soil fertility using Trichoderma bacteria and increase the absorption of nutrients by crops, and can improve the absorption of phosphoric acid by aluminum in the purified water cake. The effect of fixation was also found to be small.

[First step]
The first step is to prepare a mixture containing Trichoderma bacteria, purified water cake, and cellulose material.

(bacteria of the genus Trichoderma)
Bacteria of the genus Trichoderma are a type of filamentous fungi that are resident in soil and the like. Bacteria of the genus Trichoderma include Trichoderma virens, Trichoderma harziamun, Trichoderma hamatum, and Trichoderma viride. viride).

The Trichoderma bacteria used in the production method of the present invention have the ability to decompose cellulose. Whether or not a Trichoderma bacterium has the ability to decompose cellulose can be determined, for example, by inoculating the bacterium into a medium using cellulose as a carbon source and culturing the bacterium, and then judging from the growth status of the bacterium. Alternatively, the determination may be made based on the presence or absence of cellulase activity in a crude enzyme solution of Trichoderma bacteria. The cellulase activity of the crude enzyme solution can be calculated by subjecting the filter paper and the crude enzyme solution to an enzymatic reaction and quantifying the reducing sugars produced by the enzymatic reaction using a DNS method or the like.

The Trichoderma bacterium used in the first step is preferably derived from a bacterium that has been cultured for three months or more in soil embedded with cellulose and collected from a layer of soil in which the cellulose has been decomposed. Examples of the soil include outdoor soil such as field soil and garden soil, and specific examples include Masago soil, Kanuma soil, Akadama soil, and humus. Trichoderma bacteria are also present in these outdoor soils. By embedding cellulose in these soils and leaving it in a natural environment for a long period of time, the resident Trichoderma bacteria decompose the cellulose and multiply as a nutrient source. Trichoderma bacteria, which have a high cellulose decomposition ability among Trichoderma bacteria, can be collected from the cellulose-decomposed layer in the soil where cellulose is embedded and cultured (left) for three months or more. There is no particular limit to the period of culturing (leaving), but cellulose can be decomposed by embedding cellulose in soil and culturing it for more than 3 months (for example, 3 to 7 months) from autumn to spring (in an environment of about 0°C to 25°C). It is easy to select the bacteria collected from the collected layer, and it is easy to obtain Trichoderma bacteria with high cellulose decomposition ability.

Bacteria collected from soil can be identified to the genus level based on morphological characteristics observed under a microscope, and the species level can be identified by genetic analysis based on the base sequence information of the amplified product of ribosomal genes by PCR. Genetic analysis can be performed, for example, by PCR using DNA extracted from Trichoderma bacteria cells as a template and specific primers. DNA extraction can be performed by a conventional method, and a general DNA extraction kit can be used. For example, DNeasy Plant Mini Kit manufactured by Qiagen can be used.

The Trichoderma genus bacteria are preferably Trichoderma virens and/or Trichoderma harziamun, more preferably Trichoderma virens. These fungi have a small effect on crop growth, and tend to kill pathogenic bacteria and nematodes that cause soil diseases, and help crops absorb nutrients by decomposing organic matter in the soil.

As Trichoderma virens, for example, using DNA extracted from the cell as a template, a forward primer containing the base sequence represented by SEQ ID NO: 1 (5'-GTTGGGGATCGGCCCTTTAC-3') and SEQ ID NO: 2 (5'-CAGCGGGTATTCCTACCTGA). A bacterium containing DNA that can be amplified by PCR using a reverse primer containing the base sequence represented by -3') can be used. Such bacteria use DNA extracted from the cells of the bacteria as a template, and a forward primer containing the base sequence represented by SEQ ID NO: 1 (5'-GTTGGGGATCGGCCCTTTAC-3') and SEQ ID NO: 2 (5'- PCR is performed using CAGCGGGTATTCCTACCTGA-3'), and the presence or absence of bacteria can be determined and the number of bacteria can be calculated based on the presence or absence of fluorescence.

Specifically, determination of bacteria and calculation of the number of viable bacteria by PCR using the above forward primer and the above reverse primer can be performed as follows. This method is suitable for determining Trichoderma virens 911 bacteria (sometimes simply referred to as "911 bacteria"), which is a type of Trichoderma virens, and for determining the number of viable bacteria.

(Determination of bacteria)
(a1) Place approximately 1 g of sample (soil, soil improvement material, culture soil mixed with soil improvement material, etc.) into a test tube containing 20 mL of sterile water, stir gently, and add 0.5 mL of this suspension to Trichoderma Semi-synthetic selective medium for bacteria (composition: 1000 mL of potato decoction, 20 g of L-(-)-sorbose, 30 mg of rose bengal, 0.25 g of chloramphenicol, 20 g of agar, after autoclaving, tolclofos-methyl hydrating agent) 50 mg, 40% mepanipirim hydrating agent (0.05 mL, pH 6.8) and cultured at 25°C for 3 days.
(a2) Colonies of bacteria grown on the selective medium of (a1) above were transferred to PDA medium supplemented with chloramphenicol (composition: 1000 mL of potato broth, 25 g of dextrose, 20 g of agar, 0.25 g of chloramphenicol) under pressure. Steam sterilized, pH 7.0) and cultured at 25°C for 5 days.
(a3) From the colony formed on the chloramphenicol-added PDA medium of (a2) above, scrape off about 100 mg of the contained spore cells, and extract the bacterial chromosomal DNA from the contained bacterial cells using Qiagen's DNeasy. Extract using Plant Mini Kit.
(a4) Using chromosomal DNA extracted from cells derived from bacteria as a template, a primer for discriminating between a forward primer containing the base sequence represented by SEQ ID NO: 1 above and a reverse primer containing the base sequence represented by SEQ ID NO: 2 above Bacteria-specific PCR method is performed using It is. The composition of the PCR reaction solution was 2.5 μL of 10× reaction buffer, 2 μL of dNTPs (2 mM each), 1 μL of primers (10 μM), and 0.25 μL of Taq polymerase (2.5 units/μL) (Toyobo , Osaka, Japan), 2 μL of template DNA, and 16.25 μL of sterile water were added to make a total volume of 25 μL. The PCR reaction conditions were 95°C for 2 minutes, 39 cycles of 95°C for 30 seconds, 55°C for 50 seconds, 72°C for 1 minute, and further extended at 72°C for 5 minutes. After the PCR reaction is completed, 10 μL of the reaction solution is subjected to 3% agarose electrophoresis, and the bacteria can be identified by visually confirming the amplified DNA fragment (approximately 170 bp) by a fluorescence reaction using an ethidium bromide UV light source. is possible.

(Number of viable bacteria)
In order to monitor the number of viable bacteria in a sample (the number of spores of bacteria in soil), a diagnosis method using quantitative PCR using real-time PCR is as follows.
(b1) DNA is extracted from the sample using an ISOIL DNA extraction kit (manufactured by Nippon Gene Co., Ltd.). As primers for monitoring bacteria used in the quantitative PCR method, a forward primer containing the base sequence represented by the above SEQ ID NO: 1 and a reverse primer containing the base sequence represented by the above SEQ ID NO: 2 are used.
(b2) The composition of the primer reaction solution is 5 μL of 2×SYBR Green Supermix, 1 μL of forward primer (100 nM), 1 μL of reverse primer (50 nM), 1 μL of template DNA, and 2 μL of sterilized water, totaling 10 μL. .
(b3) The temperature reaction conditions for quantitative PCR are one cycle of 96°C for 3 minutes, followed by 49 cycles of 95°C for 20 seconds, 60°C for 25 seconds, and 72°C for 25 seconds. The dissociation curve was set in the range of 65°C to 95°C in 5 second increments of 0.5°C, and the occurrence of non-specific PCR products was confirmed. Finally, the amount of bacterial DNA (picograms) per gram of sample is quantified, and the number of spores relative to the amount of DNA is calculated.

The number of Trichoderma bacteria in the mixture is 5.0 x 10 ² cells/g (cfu/g) to 1.0 x ^{10 5} cells/g, or 1.0 x 10 ³ to 1.0 x 10 5 cells/g. They can be mixed (seeded) at a density of 1.0×10 ⁴ pieces/g. If the number of Trichoderma bacteria is too small, it is not preferable because it takes a long time to grow the number of bacteria to a predetermined amount.
For the bacteria of the genus Trichoderma, prepare a suspension containing Trichoderma in water etc. so that the amount of bacteria of the genus Trichoderma becomes a predetermined amount, and adjust the number of bacteria of the genus Trichoderma in the mixture by the amount of this suspension.・Can be managed. For example, a suspension of 911 bacteria of 1.0×10 ⁸ cells/mL to 1.0 ^{×10 10} cells/mL can be used.

(Water purification cake)
Purified water cake is made by concentrating, dewatering, and drying sediments such as earth and sand (purified water sludge, sedimented sludge) generated during the precipitation treatment and filtration treatment processes at water purification plants. Water purification cake is mainly composed of particles such as sand and clay. It may also contain a flocculant, etc. used in steps such as precipitation treatment.
Purified water cake does not inhibit the growth of bacteria, and because it is porous, Trichoderma bacteria easily adsorb to it. In addition, since water can be contained in an appropriate amount, air permeability and moisture content can be adjusted to create a growth environment in which aerobic Trichoderma bacteria can easily proliferate. For this reason, purified water cake is an excellent carrier. Furthermore, by using the purified water cake as a culture carrier for Trichoderma bacteria, the bacteria can be used as is as a soil improvement material without separating the bacteria after culturing.

The purified water cake may be a purified water cake provided from a water purification plant or a commercially available purified water cake as it is, or may be used after being sintered, crushed, classified, etc. For example, large lumps can be removed and used by sieving using a sieve with openings of 10 mm or less, 8 mm or less, or 5 mm or less.

The water content of the water purification cake depends on the drying method at the water purification plant, but is, for example, 10 to 80% by mass. Depending on the moisture content of the mixture to be prepared, the purified water cake may be used as is or after drying.

(cellulose material)
Cellulosic materials include paper, paper products, wood, sawdust, grass, cotton, and the like. These cellulose materials are preferably used after being crushed or cut, as they are easily mixed with the purified water cake.

Among cellulosic materials, paper is preferred. The paper may be general paper such as copy paper, processed paper such as laminated paper, newspaper, cardboard, etc. Even if it is unused paper, it may be waste paper (used paper). It may be. In addition, from the viewpoint of mixability and ease of decomposition, among papers, shredded paper pieces and/or flocculent materials pulverized into cotton-like materials are preferable.
Conventionally, compared to plant-based materials such as wood and sawdust, paper has been used as a material for manufacturing soil improvement materials because there is a concern that the appearance quality of the product will deteriorate if it remains without being decomposed. It was difficult to use. However, it was found that by using the manufacturing method of the present invention, in particular, by using paper strips and cotton-like materials, it was possible to decompose most of them, and the appearance quality was also good. The paper strip can have, for example, a side of 1 mm to 1 cm or 1 mm to 5 mm.

It is preferable to use waste paper as the cellulose material since waste can be effectively utilized. For example, shredded waste paper has traditionally had short fiber length, making it unsuitable for reuse as raw material for papermaking, and due to confidentiality concerns, it has usually been disposed of by incineration, so there has been little progress in recycling it. In the manufacturing method of the present invention, shredded waste paper with short fiber length is easily decomposed by Trichoderma bacteria, and it is difficult to remain in the final product (soil improvement material), making it difficult to use it as a recycled material from the viewpoint of confidentiality. can also be reduced. In addition, shredded waste paper is one of the suitable cellulose materials because it can be procured at low cost.

(Other ingredients)
The mixture may contain ingredients other than Trichoderma, purified water cake and cellulosic material. For example, as described below, water may be added to achieve a set moisture content. Additionally, clay minerals such as vermiculite, smectite, kaolinite, montmorillonite, beidellite, saponite, hectorite, nontronite, sauconite, stevensite, and mica may be included to adjust the air permeability of the mixture. For example, by adding 0.01 to 0.5 parts by mass of clay mineral to 1 part by mass of water purification cake (solid content), it becomes easier to mix water purification cake and cellulose material, and the air permeability of the mixture improves. However, bacteria of the genus Trichoderma may grow more easily.

(blend)
The mixture prepared in the first step includes Trichoderma bacteria, purified water cake, and cellulosic material.

The mixture preferably contains purified water cake and cellulose material as main components in terms of solid content. Total mass ratio of purified water cake and cellulose material to the mixture in terms of solid content ((mass of purified water cake (in terms of solid content) + mass of cellulose material) / mass of mixture (in terms of solid content) x 100 (%)) can be 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, etc. Further, the upper limit thereof can be 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 85% by mass or less, 75% by mass or less. The mass of the mixture (in terms of solid content) can be calculated as "mass of mixture x (100% - water content (%)))/100", and the mass of the purified water cake (in terms of solid content) can be calculated as "mass of mixture It can be calculated by ``mass of cake x (100% - moisture content (%)) / 100''.

Furthermore, the mass ratio of the cellulose material to the purified water cake in terms of solid content (mass of cellulose/mass of purified water cake (in terms of solid content)) is preferably 0.001 to 0.8. The lower limit is preferably 0.005 or more, more preferably 0.008 or more, and still more preferably 0.01 or more. Moreover, it is good also as 0.05 or more or 0.1 or more. The upper limit thereof is preferably 0.5 or less, more preferably 0.35 or less, and still more preferably 0.25 or less. Although it depends on the supply amount of Trichoderma bacteria, if the amount of cellulose material is too small for the purified water cake, the growth of Trichoderma bacteria will peak out earlier, and the growth of the bacteria will tend to be suppressed. Furthermore, if there is too much cellulose material in the purified water cake, a large amount of cellulose material remains without being decomposed, and the appearance quality of the soil improvement material tends to deteriorate.

There is no particular restriction on the water content of the mixture, but if the water content of the mixture is too high, the mixture will become anaerobic, and Trichoderma bacteria will not be able to grow or the growth rate will be significantly reduced. Furthermore, even if the water content of the mixture is too low, the growth rate of Trichoderma bacteria tends to decrease. The water content of the mixture (mass of water/mass of mixture x 100 (%)) is preferably 50% by mass or less, and may be 45% by mass or less, 40% by mass or less, or the like. Moreover, the lower limit can be 10% by mass or more, 20% by mass or more, 30% by mass or more, etc. Depending on the moisture content of the purified water cake used, the purified water cake may be dried, water may be added to the purified water cake, or the concentration of the Trichoderma-containing suspension may be adjusted so that the prepared mixture has the set moisture content. All you have to do is make it happen.

Among these, the first step is preferably a step of preparing a mixture containing Trichoderma bacteria, purified water cake, and waste paper. As a result, the waste material is effectively used as a carrier and a nutrient source for Trichoderma bacteria, thereby reducing the cost of raw materials and making it possible to produce the product at low cost.

(Mixing method)
Although there is no particular restriction on the order in which Trichoderma bacteria, purified water cake, and cellulose material are mixed, it is preferable to mix Trichoderma bacteria after mixing purified water cake and cellulose material. Trichoderma bacteria are usually supplied as a suspension, so if you mix Trichoderma bacteria and purified water cake and then mix the cellulose material, lumps tend to form in the mixture, making it difficult to mix uniformly. It's hard to do. Furthermore, when Trichoderma bacteria and cellulose material are mixed together and then mixed with a purified water cake, lumps tend to form in the mixture, making it difficult to mix uniformly. Incidentally, the occurrence of lumps can also be suppressed by adjusting the water content of the suspension of Trichoderma bacteria.

Although the purified water cake and the like also contain bacteria of the genus Trichoderma and other bacteria, the mixture is prepared by separately adding bacteria of the genus Trichoderma as useful bacteria to the purified water cake and the cellulose material. It is preferable that the useful bacteria added to the purified water cake and the cellulose material are substantially only Trichoderma bacteria.

[Second step]
The second step is a step of culturing Trichoderma bacteria in the mixture prepared in the first step.

The culture temperature in the second step is not particularly limited as long as it is a temperature at which Trichoderma bacteria can proliferate. The culture temperature is preferably 40°C or lower, more preferably 35°C or lower, and even more preferably 30°C or lower. Further, the culture temperature is preferably 15°C or higher, more preferably 25°C or higher.

The cultivation period in the second step is changed as appropriate depending on the temperature and the initial number of bacteria, but if the number of Trichoderma bacteria is 1.0 x 10 ⁵ cells/g or more or 1.0 x 10 ⁸ cells/g It is preferable to carry out the treatment until the amount reaches /g or more. The number of bacteria in the soil improvement material may be calculated using the above PCR method or by back calculation from the number of colonies as described later in Examples. The culture period is, for example, preferably 5 days or more, more preferably 10 days or more, and even more preferably 20 days or more. The upper limit is not particularly limited, but may be 60 days or less, 40 days or less, etc.

During the cultivation period of Trichoderma bacteria, a blower or the like may be used to maintain a well-ventilated environment in order to maintain a predetermined temperature. In addition, in order to prevent the mixture from drying out, watering or the like may be performed from time to time.

For example, the mixing in the first step can be performed using a stirrer or the like. In the second step, the mixture prepared using a stirrer or the like is placed in a breathable container such as a flexible container bag, and left to stand at a predetermined temperature, or is further divided into small bags and left to stand. . Thereby, Trichoderma bacteria can be cultured and grown. The small bag may be a soil bag used for storing commercially available soil. Even without using bags made of materials with higher air permeability or without carefully controlling air permeability, the soil improvement material obtained by the manufacturing method of the present invention can be piled up by making holes in several places in the soil bag. Even when stored, the number of bacteria can be maintained stably.

<Soil improvement material of the present invention>
The soil improvement material obtained by the production method of the present invention (hereinafter sometimes referred to as "soil improvement material of the present invention") contains Trichoderma bacteria as a predominant species. The number of Trichoderma bacteria in soil improvement materials is 1.0×10 ⁵ cells/g to 1.0× ^{10 9} cells/g, or 1.0×10 ⁷ cells/g to 1.0×10 It can be ⁸ pieces/g. The number of bacteria may be calculated using the above-mentioned PCR method or by back calculation from the number of colonies as described later in Examples.
The soil improvement material of the present invention uses Trichoderma bacteria to improve soil, so it can cause problems that can occur when using chemical pesticides (adverse effects on humans, livestock, and the natural environment, chemical damage to crops, and pathogenic bacteria). drug resistance, etc.) are less likely to occur.

<Soil improvement method>
Soil can be improved using the soil improvement material of the present invention. The soil improvement material of the present invention is used by mixing with target soil. For soil improvement materials, the concentration of Trichoderma bacteria is measured, and the concentration of Trichoderma bacteria in the soil at the time of application of the soil improvement material is 1.0 x 10 ⁴ cells/g to 1.0 x 10 ⁶ cells/g. It is preferable to apply it in an amount of 5.0×10 ⁴ pieces/g to 5.0× ^{10 5} pieces/g.
The soil improvement material of the present invention may be applied directly to the soil in a field, or the soil improvement material may be mixed with farming soil such as agricultural soil or gardening soil, and then the soil may be used to cultivate crops.
In addition, in order to ensure that Trichoderma bacteria in the soil improvement material spreads sufficiently into the soil, after mixing the soil improvement material and the soil, leave it for a certain period of time (for example, 10 days or more or 20 days or more), and grow the crops. May be used for cultivation. In particular, if the soil improvement material is allowed to stand still for 30 days or more, Trichoderma bacteria in the soil improvement material can be used in a state where it has spread throughout the soil.

The soil improvement material of the present invention can be used to promote crop growth and improve germination rates. Crops are not particularly limited, but include leafy vegetables such as Komatsuna, spinach, Chinese chrysanthemum, green onions, celery, salad greens, bok choy, Chinese cabbage, cabbage, and broccoli, fruit vegetables such as tomatoes, watermelons, strawberries, cucumbers, and eggplants, daikon radish, and carrots. , potatoes, burdock, and other root vegetables.

The soil improvement material of the present invention can be used to suppress continuous cropping damage. It may also be used to control plant diseases such as root rot wilt and anthracnose.

(An example of soil improvement method)
c1) Mix the soil improvement material and commercially available soil at a ratio of 1:80 to 1:120 (kg/kg).
c2) Stir the soil mixed with soil improvement materials using a concrete mixer for 30 to 1 hour. If any lumps occur, loosen them.
c3) After leaving the soil mixed with soil improvement materials for about one month, it is used for cultivating crops.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is changed.

<Raw materials used>
The water purification cake used was produced by Nagato Co., Ltd. (granular baked product under 4 mm, sieved after heat sterilization in a kiln, specific gravity 0.8 to 0.85 kg/L, water content 34.3% by mass). .

As the mineral clay (other components), fine vermiculite grains (vermiculite from Hebei Province, China/fired by Oishi Bussan) (size approximately 1 mm) were used.

Paper fluff and shredded waste paper were used as cellulose materials. The paper fluff is made by cutting high-grade fusuma paper with a thickness of 5 mm into 1 cm width pieces, finely pulverizing the bran chips into chips using a shredder, and then using a high-speed pulverizer to make the paper fluff. As the shredded waste paper, strips of 1 mm x 5 mm or 1 mm x 9.6 mm were used.

Trichoderma virens 911, a type of Trichoderma virens, was obtained from the Kyushu University Tropical Agricultural Research Center and used as the Trichoderma genus. A suspension of Trichoderma virens 911 bacteria in water (Trichoderma bacteria solution) was prepared and used at a concentration of 10 ⁹ conidia per mL (10 ⁹ conidia/mL). .

The Trichoderma virens 911 bacterium was cultivated for about half a year in soil embedded with shredded waste paper (1 mm x 5 mm) crushed with a shredder, and bacteria collected from the soil in the layer where the shredded waste paper had been decomposed were selectively cultured on a medium. However, other bacteria have been removed through pure culture, resulting in a single type of bacteria.
Specifically, a hole approximately 25 cm long, 25 cm wide, and 30 cm deep was dug in outdoor soil (masago soil), and shredded waste paper (1 mm x 5 mm) was placed in the hole to a depth of approximately 10 cm. Furthermore, after about half a year (from autumn to spring) after covering the soil with soil, the covered soil was dug to a depth of 20 cm and bacteria were collected from the layer of soil where the shredded waste paper had been sufficiently decomposed. This bacterium was selectively cultured on a medium, other bacteria were removed by pure culture, and a single species was preserved.

This Trichoderma virens 911 bacterium has the following properties.
(A1) Has cellulose decomposition ability.
(A2) Using DNA extracted from cells as a template, a forward primer containing the base sequence represented by SEQ ID NO: 1 (5'-GTTGGGGATCGGCCCTTTAC-3') and SEQ ID NO: 2 (5'-CAGCGGGTATTCCTACCTGA-3') It contains DNA that can be amplified by PCR using a reverse primer containing the indicated base sequence.

<Reference example>
(1) Evaluation by PCR method Figure 1 shows the electrophoretic profiles of PCR products obtained by PCR using the above primers for Trichoderma virens 911 bacteria and four other bacteria (all obtained from Kyushu University Tropical Agriculture Research Center). show. As shown in FIG. 1, fluorescence of Trichoderma virens 911 bacteria was confirmed.
Furthermore, FIG. 2 shows the results of evaluating the sensitivity of qPCR using a series of 10-fold dilutions (4×10 ¹ to 4×10 ⁻⁵ ng) of the genomic DNA of Trichoderma virens 911 extracted from the culture solution. Figure 2(a) is a standard curve based on the relationship between cycle threshold and Trichoderma virens DNA concentration, Figure 2(b) is a melting curve (fluorescence vs. temperature), and Figure 2(c) is a standard curve based on the relationship between cycle threshold and Trichoderma virens DNA concentration. 911 is a real-time amplification curve of DNA of the Bacterium virens 911. As shown in FIG. 2, it can be seen that Trichoderma virens 911 has high sensitivity in qPCR using the above primers.

(2) Bacterial culture test A culture test was conducted using two strains of each of the three species of bacteria.
・Test bacterial strains (all obtained from Kyushu University Tropical Agriculture Research Center)
Trichoderma virens 211 bacteria, 911 bacteria Trichoderma harzianum 32B bacteria, 1511 bacteria Clonostachys rosea 811 bacteria, 1523 bacteria / Method Each strain was precultured on a PDA plate medium and beaten with a cork borer with a diameter of 9 mm. I pulled it out. This was placed on the edge of a new PDA plate medium and cultured for 7 days at a temperature of 5°C to 35°C, and the length of hyphae of each bacterial strain after culture was measured at each culture temperature.
-Results As shown in Figure 3, Trichoderma bacteria showed good results.

(3) Cellulase activity Trichoderma virens 911 bacteria, Trichoderma harzianum 32B bacteria, and Clonostachys rosea 811 bacteria were each cultured on a PDA plate medium (25°C, in the dark for 8 days), and the medium was punched out with a cork borer to a diameter of 10 m. m disk Obtained.
Carboxymethyl cellulose sodium salt: 20g, (NH ₄ ) ₂ HPO ₄ : 1.5 g, KH ₂ PO ₄ : 0.46 g, K ₂ HPO ₄ : 1.0 g, MgSO _{4.7H 2} O: 0.5 g, Thiamine hydrochloride A culture medium with a composition of: 500 μg/L, water: 1000 mL (medium pH after sterilization: 7.4) was prepared, and 100 mL was dispensed into a 500 mL flask and sterilized under high pressure for 20 minutes. This medium was inoculated with 10 mm diameter disks of each of the above bacteria. Next, shaking culture was carried out in the dark at 30° C. for 3 to 13 days (reciprocating shaking at 110 rpm), and the bacterial cells were filtered off, and the obtained filtrate was used as a crude enzyme solution.
Add 1.0 mL of 0.1M buffer (pH 4.8, pH 6.0, pH 9.5) and 0.5 mL of crude enzyme solution to a filter paper (Whatman (registered trademark) No. 1, approximately 50 mg) with a width of 1 cm and a length of 6 cm. was added and the enzymatic reaction was carried out at 50°C for 60 minutes. As for the 0.1M buffer, an acetate buffer was used for pH 4.8, a phosphate buffer was used for pH 6.0, and a carbonate buffer was used for pH 9.5. Next, 4 mL of DNS solution was added, the mixture was left at 100° C. for 5 minutes, and 9.5 mL of pure water was added. Thereafter, the absorbance at 540 nm was calculated. For the blank, pure water was used instead of the crude enzyme solution. The measurement was repeated twice.
Table 1 shows the ratio of absorbance of each bacteria to Trichoderma harzianum 32B bacteria ((absorbance when crude enzyme of each bacteria is used - absorbance of blank)/(absorbance when crude enzyme solution of 32B bacteria is used - absorbance of blank) absorbance)). The value was the average value of two measurements.

<Manufacture example 1>
The purified water cake and paper fluff were mixed in a ratio of 10:1 (mass ratio) (4:1 (volume ratio)), distilled water was added to give a water content of 40%, and the mixture was autoclaved. Trichoderma bacteria solution was added to the mixture of the purified water cake and the paper fluff to a concentration of 1000 cfu/mL of conidia, that is, 1000 conidia per mL, and the purified water cake and the paper fluff were A mixture containing Trichoderma and Trichoderma was obtained. This mixture was cultured at 30° C. for 4 weeks to obtain soil improvement material 1 (soil supplement 1).

The concentration of conidia in the soil improvement material was measured every week of culture. The concentration of conidia was calculated as the number of conidia in 1 g of the mixture. To measure the number of conidia, suspend 1 g of the mixture in 10 mL of sterilized water, apply 100 μL of the 10-fold and 100-fold diluted solutions on a PDA plate medium, and after culturing at 30°C for 5 days, grow. The number of colonies that arrived was actually measured, and the number of spores (concentration) was calculated by back calculation from the number of colonies.
As shown in FIG. 4, the number of spores increased rapidly in the first week from an inoculum concentration of 1000 cfu/mL. It was also confirmed that they remained stable for a long period of time.

<Manufacture example 2>
[Manufacture example 2-1]
Shredded waste paper (1 mm x 5 mm): purified water cake: vermiculite = 1:8:1 (mass ratio) was mixed in a 200 mL Erlenmeyer flask, and after steam pressure sterilization in an autoclave, the bacterial spore density was Trichoderma bacteria solution was added to the solution at a concentration of 10 ³ cfu/mL to prepare a mixture. This mixture was cultured at 30° C. for 40 days to obtain soil improvement material 2 (soil supplement 2).

[Production example 2-2]
Soil improvement material 3 (soil supplement 3) was obtained in the same manner as in Production Example 2-1 except that shredded waste paper (1 mm x 5 mm) was replaced with shredded waste paper (1 mm x 9.6 mm).

Comparing the number of bacterial spores in soil improvement material 2 and soil improvement material 3, the number of bacterial spores in soil improvement material 3 tended to be higher after one week of culturing the mixture, but after 20 days or more of culture, soil improvement material 2 had a higher number of spores. The number of spores of these bacteria increased. After 40 days of culture, Trichoderma bacteria had grown to about 3.5 x 10 ⁶ cfu/g in soil improvement material 2, and to about 4.0 x 10 ⁵ cfu/g in soil improvement material 3.

FIG. 5 shows the condition of the Erlenmeyer flask in which Trichoderma bacteria were cultured and the condition of the soil improvement material taken out after 40 days of culture. As shown in the photo in Figure 5, as a result of taking out the inside, the shredder paper has been decomposed, so there is little possibility that the remains (residue) of the shredder paper will remain in the soil improvement material obtained. It was suggested that if a certain high concentration of Trichoderma bacteria is used in the process, the quality of the product will not be affected. In particular, paper strips cut to 1 mm x 5 mm had almost no undecomposed debris remaining.

<Manufacture example 3>
Purified water cake (3000 L) and vermiculite (300 L) were placed in a large stirrer. Shredded paper (1 mm x 5 mm, 24 kg) was added while stirring, and the mixture was further stirred for 5 minutes. A solution prepared by diluting Trichoderma fungus solution (1 L) with water (100 L) was showered here, and the mixture was further stirred for 10 minutes. The mixture (approximately 3300 L, water content 37.3% by mass) was discharged into three flexible container bags (flexible containers).
The mixture in the flexible container was packed into a 20L bag. This was stored in a warehouse (25° C. to 30° C.) for about one month, and was used as soil improvement material 4 (soil supplement 4).

<Plant cultivation test (I)>
The following four trial soils were produced.
・“Agricultural soil (untreated)”
Only agricultural soil (soil supplement 1 not applied).
・"Gardening soil (untreated)"
Only horticultural soil (soil supplement 1 not applied).
・"Agricultural soil (processing)"
Soil Supplement 1: Agricultural soil = 1:100 (by weight) of culture soil, which was mixed and left in a dark place at 15°C for one month.
・"Gardening soil (processed)"
Soil Supplement 1: Horticulture soil = 1:100 (by weight) of potting soil that was mixed and left in a dark place at 15°C for one month.
In addition, in the culture soil to which Soil Supplement 1 is applied, leaving it for one month is based on the assumption that it takes about one month for Trichoderma bacteria to spread throughout the soil in agricultural or horticultural soil. This is to encourage sufficient proliferation (growth) of bacteria.
In addition, as agricultural soil, 22 wt% of beet moss, 30 wt% of prepared beets, 37 wt% of vermulite, 2 wt% of bamboo charcoal, and 9 wt% of baked soil were used. The gardening soil used was 15 wt% beet moss, 20 wt% rag soil, 10 wt% vermiculite, 18 wt% roasted rice, 22 wt% bark, and 15 wt% barite.
Cultivating soil test plots were prepared using the four trial cultivating soils prepared, and a plant cultivation test was conducted.

(1-1) Komatsuna plant cultivation test Put 2L of trial soil in a No. 9 unglazed pot, place 30 seeds of Komatsuna (variety: round leaves) at equal intervals on the soil surface in the pot, and pour the same trial soil on top. Cover. After seeding, the pots were brought to the Kyushu University Biological Environment Utilization Center's artificial weather equipment (phytotron facility) and cultured for 4 weeks at room temperature (minimum temperature 15°C, maximum temperature 20°C or higher, sunlight naturally controlled). did. The germination rate of Komatsuna seeds was compared in the test plots using four trial soils. Furthermore, 4 weeks after sowing, the total length and dry weight of the grown Komatsuna were measured and compared within the test plots using the four trial soils.

Table 2 shows the germination rate of Komatsuna seeds for each trial culture soil. Compared to gardening soil, farm soil tended to have a lower germination rate for Komatsuna seeds. Furthermore, when comparing plots treated with Trichoderma bacteria and plots that were not treated, there was a tendency for the germination rate to be higher in soil inoculated with useful bacteria. This led to the possibility that Trichoderma may be involved in promoting the germination of inoculated Komatsuna seeds.

FIG. 6 is a photograph of Komatsuna seedlings one week and three weeks after sowing. As can be seen from the photograph in Figure 6, there was a clear difference in the growth of Komatsuna between the test plots treated with soil improvement materials (sa treatment) and the untreated test plots.

Table 3 shows the average values of plant length and dry weight of Komatsuna seedlings 4 weeks after sowing of Komatsuna. No statistically significant difference in plant length was observed between test plots to which soil improvement materials were applied (Sa treatment) and untreated test plots, or between soil types (cultivating soil for farmers and cultivating soil). On the other hand, with regard to dry weight, clear differences were observed depending on the difference in the trial soil. In particular, the data from the test plots to which soil improvement materials were applied (Sa treatment) and the untreated plots showed that the treated plots had 1.5 to 1.7 times higher data, indicating that the soil improvement material treatment was 1.5 to 1.7 times higher. It was revealed that this method had a remarkable effect on increasing the dry weight of young Komatsuna seedlings.

(1-2) Cultivation test of tomato seedlings Tomato seeds (variety Matina) were sown in unglazed pots, and seedlings with 4 to 5 leaves developed were produced. The produced tomato seedlings were transplanted in groups of 5 to 30 cm long planters and grown at room temperature of 20° C. in the above-mentioned phytotron facility.

FIG. 7 shows the results of a growth test of tomato seedlings after one month. As shown in Figure 7, in the treated plots where soil improvement materials were added, the total length increased by 10 to 20% and the dry weight increased by 1.3 to 1.6 times in both agricultural soil and horticultural soil. Regarding seedlings, the effect of promoting plant growth was also shown, similar to the test results for Komatsuna.
This result proves that the addition of soil improvement materials to the soil is very effective in improving the germination rate of plant seeds and the growth of seedlings. It can also be expected to increase the production of crops such as fruits.

A comparison of the amount of absorption of fertilizer elements (nitrogen, phosphoric acid, and potassium) by Komatsuna in the test plot treated with soil improvement materials and Komatsuna in the untreated test plot showed that Komatsuna in the test plot treated with soil improvement materials did not absorb much fertilizer. The amount of absorption of elements increased by about 1.5 to 3 times. In addition, the amount of fertilizer absorbed by tomatoes in the test plot treated with the soil improvement material increased by about 1.5 times compared to the untreated test plot.

<Plant cultivation test (II)>
Four trial culturing soils were prepared in the same manner as in Plant Cultivation Test (I) except that Soil Supplement 2 was used instead of Soil Supplement 1 as a soil improvement material. Cultivating soil test plots were prepared using the four trial cultivating soils prepared, and a plant cultivation test was conducted.

(2-1) Cultivation test of radish The growth of seedlings was investigated one month after sowing. The results are shown in Table 4 and FIG. In particular, the effect of enlarging rhizomes was remarkable when soil improvement materials were applied. In both agricultural soil and gardening soil, trial culture soil mixed with Soil Supplement 1 showed an increase in leaf stem length of 10% or more and an enlargement of radish tissue (rhizome) by 50% or more. Therefore, it was suggested that soil improvement materials may be very suitable for growing root vegetables such as radish.

(4) Okra cultivation test After planting the seedlings, the total length and average fruit weight of the seedlings were measured for up to 2 months. Table 5 shows the results. Regarding the average length, there was no significant difference in the effect of the trial cultivation soil with the addition of soil improvement materials in both agricultural soil and gardening soil, but regarding the average fruit weight, the effect of the trial cultivation soil by adding soil improvement materials was 15% in both agricultural soil and gardening soil. It was found that the soil improvement materials had an effect on the increase in fruit yield.

(5) Green peppers For green peppers, data on the total plant length and average fruit weight were collected for up to about 40 days after planting the seedlings. Regarding the total plant length, the results are shown in Table 6. No effect of soil improvement materials was observed, and no clear difference was observed between agricultural soil and horticultural soil. However, the effect of soil improvement materials was remarkable for average fruit weight, with an increase of about 15% in agricultural soil and 23% in gardening soil. Even though the crops were planted using planters and no additional fertilizers were applied, it was suggested that by applying soil improvement materials, yields could be expected to increase to the same level as in open field cultivation.

(6) Celery Figure 9 shows the results of a celery growth test. In the celery growth test, it was confirmed that the roots had good root tension.

<Plant cultivation test (III)>
(3-1) Cultivation test of salad greens Using the following soil improvement materials, the growth of salad greens (provided by Fukuoka Engei Co., Ltd.) was evaluated using a greenhouse manufactured by Oishi Bussan Co., Ltd. as a test site.

(Soil improvement materials used)
・Soil improvement material: soil supplement 4
・Comparative soil improvement material 1: Bacterial black juice continuous cultivation obstruction block W (manufactured by Yasaki Co., Ltd.) (Soil in which zeolite is impregnated with Yasaki's product microbial material "Bacterial black juice (registered trademark)" (photosynthetic bacteria) improved materials)
・Comparative soil improvement material 2: Soil improvement master (manufactured by Natural Applied Science Co., Ltd.) (soil improvement material made by impregnating vermiculite with microbial material "VS bacteria")
・Comparative soil improvement material 3: Fertilizer retention capacity (manufactured by Komeri Co., Ltd.) (granular soil improvement material whose main component is humic acid)
・Comparative soil improvement material 4: Molden Rensaku-kun (Tobima Co., Ltd.) (soil improvement material with 100% zeolite component)

(Growth evaluation method)
Seedlings of salad greens to be planted were raised in cell trays in advance (using seeding soil sold by Oishi Bussan Co., Ltd.).
In addition, a part of the field was divided into 18 6 x 3 plots, and appropriate amounts of 5 types of soil improvement materials were applied to 3 plots of each type so that they were not covered in the same rows and columns. No soil improvement materials were applied to three plots.
Next, 5 to 6 salad greens were planted in each plot. Additional fertilizer and pesticide spraying were performed as appropriate, and the crops were harvested 40 to 50 days later. Crop weight was measured at harvest. The weight of each plot was compared and the degree of growth was compared.

The results are shown in FIG. As shown in FIG. 10, salad greens grown in soil to which soil supplement 4 was applied showed an increase in crop weight.

(3-2) Chinese cabbage cultivation test Using soil improvement material (soil supplement 4) and comparative soil improvement material 1, growth of Chinese cabbage was evaluated.
Chinese cabbage grown in soil treated with soil improvement materials was harvested on the 70th day. All the Chinese cabbage were heads. Chinese cabbage grown in soil using comparative soil improvement material 1 was harvested on the 90th day. Half of the Chinese cabbage seeds did not form. In addition, in the soil where no soil improvement material was applied, all the Chinese cabbage did not form heads, so no harvesting was performed.
FIG. 11 is a diagram showing a photograph of Chinese cabbage on the 50th day of planting and the average weight of the edible portion of Chinese cabbage grown in each soil. As shown in FIG. 11, it was confirmed that the yield of Chinese cabbage increased in the soil to which the soil supplement was applied.

(3-3) Cultivation test of carrots, etc. Carrots, potatoes, bok choy, and broccoli were also evaluated for growth using soil improvement material (soil supplement 4) and comparative soil improvement material 1. The results are shown in FIGS. 12 and 13. As shown in FIGS. 12 and 13, it was confirmed that yields of carrots, potatoes, bok choy, and broccoli increased in the soil to which the soil supplement was applied.

<Evaluation test of soil disease suppression effect>
(4-1) Evaluation of the inhibitory effect on tomato root rot wilt (I)
Since Trichoderma fungi are mycoparasitic, using other filamentous fungi as a food source, they can be expected to be effective in suppressing the proliferation of pathogenic bacteria in the soil. Therefore, in order to verify the disease control effect of tomato root rot wilt (pathogen name: Fusarium fungi), which is an important soil disease of tomatoes, we first tested 1,000 spores of the pathogen Fusarium fungus in gardening soil or agricultural soil. Contaminated soil was prepared by mixing it so that Next, the contaminated soil was placed in a pot, and soil supplement 2 (Trichoderma bacteria added) was mixed with the contaminated soil after one month to prepare contaminated soil (pathogen inoculation + soil supplement mixed). Tomato seedlings were grown in this soil and the development of disease in the tomato seedlings was observed. As a control, tomato seedlings were grown in gardening soil or agricultural soil (soil not inoculated with pathogens) and contaminated soil without soil supplement treatment (soil inoculated with pathogens), and the development of disease in tomato seedlings was observed.
Since it takes about one month for the disease to develop, the disease state of the tomato seedlings was observed one month after transplantation to verify the suppressive effect on tomato root rot wilt.

The results are shown in Table 7 and FIG. 14. As shown in Table 7 and FIG. 14, it was proven that the soil improvement materials had a suppressive effect on tomato soil diseases. The average total length of tomatoes was 28 cm in non-pathogen-inoculated soil (healthy), while it was 17 cm in soil inoculated with pathogens, and tomato growth was suppressed by the tomato root rot wilt fungus, but soil improvement in contaminated soil In the treatment area where the material was added, growth was observed to be 41 cm, which was more than 20% faster than that of healthy individuals.
The results of this test revealed that the soil improvement material has the ability to inhibit continuous cropping damage caused by soil diseases such as tomato root rot wilt, and also to promote plant growth, indicating that multiple synergistic effects can be expected. has been proven.

(4-2) Evaluation of the suppressive effect on tomato root rot wilt (II)
Three test plots were set up: contaminated soil, contaminated soil mixed with soil improvement material 2 (contaminated soil + supplement), and gardening soil mixed with soil improvement material 2 (non-contaminated soil + supplement), and one week after sowing. Seedlings were planted, and the number of Trichoderma bacteria and soil pathogenic bacteria in each test soil was measured every week. FIG. 15 shows photographs of tomatoes grown in each soil 30 days after planting, and the results of plotting the number of bacteria against the number of cultivation days.

Root rot wilt, a continuous cropping disease of tomatoes, causes severe death in contaminated soil, but it is clear that the damage caused by the death can be reduced by applying soil improvement materials. It was also found that the tomato root rot wilt fungus maintains the number of fungi (10 ⁶ to 10 ⁷ spores/g) that enables the onset of the disease in the soil until 30 days after cultivation. Trichoderma bacteria proliferated stably until the 15th day of cultivation, and maintained a bacterial count higher than the inoculation concentration (10 ⁴ spores/g) until the 30th day of cultivation. In contaminated soil to which soil improvement materials were applied, the number of bacteria reversed within 5 days of cultivation, and it was revealed that Trichoderma bacteria strongly suppressed root rot wilt bacteria until 30 days of cultivation.

(4-3) Evaluation of the effect of suppressing soybean anthracnose Contaminated soil infected with soybean anthracnose, contaminated soil mixed with soil improvement material 2 (contaminated soil + supplement), and soil improvement material 2 were mixed. Three test plots of horticultural soil (uncontaminated soil + supplements) were set up, seeds were sown directly, and the number of Trichoderma bacteria and soil pathogenic bacteria in each test soil was measured every week. FIG. 16 shows photographs of soybeans grown in each soil 30 days after planting, and the results of plotting the number of bacteria against the number of cultivation days.

Anthracnose, a continuous cropping disease of soybean, causes severe death in contaminated soil, but it was found that death can be avoided by applying soil improvement materials. It was found that the soybean anthracnose bacteria maintains the number of bacteria (10 ⁶ to 10 ⁷ spores/g) that enables the onset of the disease until 30 days after cultivation. Trichoderma bacteria increased steadily until 10 days after cultivation, and although it decreased slightly after 15 days, the number of bacteria remained at 10 ⁵ spores/g until 30 days. By applying soil improvement materials to contaminated soil, the number of bacteria reversed after three days of cultivation, indicating that Trichoderma bacteria tended to stably suppress the growth of anthracnose bacteria.

<Continuous cultivation test>
Using Soil Supplement 2 as a soil improvement material, four trial culturing soils were used to prepare cultivating soil test plots and conducting tests.
After carrying out a cultivation test for the growth of germinated Komatsuna seedlings, we continued to cultivate Komatsuna seeds using the same soil, and conducted a continuous cultivation test to investigate the effect of Trichoderma bacteria in soil improvement materials on the continuous cultivation of Komatsuna. did.
The test was conducted in a test plot in which Komatsuna was grown using soil (horticultural soil and agricultural soil) mixed with soil supplements, and in the second test, Komatsuna was cultivated continuously without adding any additional fertilizer to the soil. did. In addition, in order to investigate the residual effect of Trichoderma bacteria in soil supplements, the density of fungal spores in the soil was measured. In addition, cultivation evaluation of Komatsuna was measured using the average value of biological weight as data. Table 8 shows the results.

As shown in Table 8, regarding the residual effect of Trichoderma bacteria in the soil supplement during continuous cultivation, a decrease of about 20 to 30% of Trichoderma bacteria was observed through continuous cultivation. It was inferred that this is probably due to the fact that pot cultivation caused Trichoderma bacteria to be washed away, resulting in a decrease in bacterial density in the soil that exceeded their proliferation. Furthermore, due to continuous cultivation, the second Komatsuna had a pale green color overall, and its biological weight had decreased by about 30 to 40% (Figure 17). This suggested that growth failure may be caused by lack of fertilizer.
However, the biological weight of agricultural and horticultural soils to which soil supplements had been added was approximately 15 to 20% higher. It was proven that there was no negative impact and that the residual effect was sufficiently ensured.
From the above points, it can be concluded that the soil supplement has extremely high safety in the plant cultivation environment due to its residual effects and runoff outside the cultivation area.

By using the soil improvement material of the present invention, soil can be improved to prevent or improve continuous cropping disorders and promote crop growth. This improves crop yields.

Claims (5)

a first step of preparing a mixture comprising a Trichoderma bacterium, a purified water cake, and a cellulose material, and a second step of cultivating the Trichoderma bacterium in the mixture;
A method for producing a soil improvement material, wherein the Trichoderma genus bacteria is Trichoderma virens. The manufacturing method according to claim 1, wherein the cellulosic material is paper. The manufacturing method according to claim 1 or 2, wherein the mass ratio of the cellulose material to the purified water cake in terms of solid content is 0.001 to 0.8. Any one of claims 1 to 3, wherein the Trichoderma bacterium used in the first step is a bacterium that has been cultured for three months or more in soil embedded with cellulose and collected from a layer of soil in which the cellulose has been decomposed. The manufacturing method described in. A soil improvement method for improving soil using a soil improvement material produced by the production method according to any one of claims 1 to 4.